Therapeutic application of carvacrol: A comprehensive review

Abstract Carvacrol is a major natural constituent and is significantly present as an essential oil in aromatic plants and is well known for its numerous biological activities. Therapeutic properties of carvacrol have been demonstrated as anti‐oxidant, anticancer, diabetes prevention, cardioprotective, anti‐obesity, hepatoprotective and reproductive role, antiaging, antimicrobial, and immunomodulatory properties. The carvacrol biosynthesis has been mediated through mevalonate pathway. Carvacrol has the anticancer ability against malignant cells via decreasing the expressions of matrix metalloprotease 2 and 9, inducing apoptosis, enhancing the expression of pro‐apoptotic proteins, disrupting mitochondrial membrane, suppressing extracellular signal‐regulated kinase 1/2 mitogen‐activated protein kinase signal transduction, and also decreasing the phosphoinositide 3‐kinase/protein kinase B. It also decreased the concentrations of alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase, and gamma‐glutamyl transpeptidase as well as also restored liver function, insulin level, and plasma glucose level. Carvacrol also has been found to exert antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Coagulase‐negative staphylococcus, Salmonella spp., Enterococcus sp. Shigella, and Escherichia coli. The current review article summarizes the health‐promoting perspectives of carvacrol through various pathways.


Biological activities of carvacrol have been shown in different in
vivo and in vitro studies including anti-oxidant, antiseptic, anticarcinogenic, anti-inflammatory, antidiabetes role, immunomodulatory, antimicrobial activity, antispasmodic, antibacterial, and growth promoter. As it is a natural cymene derivative, it has potent bacterial inhibiting abilities and due to its flavoring property used in food industry as a preservative (Churklam et al., 2020;Memar et al., 2017;Mousavi et al., 2020;Rezvi & Roy, 2019;Scaffaro et al., 2020;Sun et al., 2020). Ezz-Eldin et al. (2020) showed the antiproliferative, antiinflammatory, and pain-relieving properties of carvacrol against bronchial asthma; bronchial asthma in animals was induced by an intranasal dose of ovalbumin. IN serum absolute eosinophil count (AEC) and immunoglobulin E (IgE) and inflammatory biomarkers like IL-3, IL-4, IL-5, IL-13, TNFα, IFNγ, and iNOS were determined in bronchoalveolar lavage fluid. The level of oxidative stress biomarkers was also measured. Results determined that carvacrol is a significant anti-oxidant and antiproliferative agent (Ezz-Eldin et al., 2020).
Plants synthesized carvacrol via the mevalonate pathway. Glucose is first decarboxylated and acetylated to acetyl coenzyme A (acetyl CoA), which could then be turned into mevalonic acid. Aromatization converts mevalonic acid to gamma-terpinene, which is then converted to p-cymene. Carvacrol was produced by the hydroxylation of p-cymene, as shown in Figure 1.

| Anti-oxidant property
Oxidative stress is caused by the imbalance between oxygen reactive species and detoxification of the reactive intermediates via biological system ability; these free radicals damage different body cell molecules such as protein, lipids, and nucleic acids. Essential oils found in plants are natural anti-oxidants that reduce cell damage caused by reactive species and prevent mutagenic and carcinogenic processes. Carvacrol has remarkably higher anti-oxidative and hepatoprotective properties, which improves the activity of enzymatic anti-oxidants (catalase, superoxide dismutase, and glutathione peroxidase) and the levels of nonenzymatic anti-oxidants (vitamin C, reduces glutathione and vitamin E) (Gursul et al., 2019;Tohidi et al., 2020). Origanum ones L. contains the highest amount of carvacrol and showed significant anti-oxidant and anticarcinogenic activities against the triple-negative breast cancer MDA-MB-231 cell line than the human glioblastoma U87 cell line (Alagawany et al., 2015; Baranauskaite et al., 2017;Sharifi-Rad et al., 2018).
The presence of hydroxyl group (OH) in carvacrol is the major reason for its radical scavenging activity (superoxide radicals, nitric oxide, and hydrogen peroxide). Its weak acid character facilitates hydrogen atoms donation to unpaired electrons, making another radical stabilized by electron scattering produced at molecule resonance structure (Cocolas et al., 2019;Mir et al., 2020). Carvacrol protects against oxidative stress made by restraint stress damages the brain, liver, and kidney. In the liver, acute pancreatitis leads to multiple organ dysfunction, and carvacrol has been investigated to have anti-oxidative and hepatoprotective properties (Guarda et al., 2011;Mir et al., 2020;Rezvi & Roy, 2019;Samarghandian, Azimi-Nezhad et al., 2016;. A study conducted by Bakır et al. in 2016 analyzed the hepatoprotective effects of carvacrol on acute pancreatitis produced by cerulein and also explored the underlying mechanism. The rats were randomly divided into two groups (1) with no therapy, (2) provided with 50 μg/kg cerulean, (3) (50, 100, and 200 mg/kg) carvacrol, and (4) cerulein + carvacrol. Carvacrol decreased pancreatitis-induced MDA and 8-OH-dG levels, and the activities of the liver SOD, CAT, and GSH-Px increased. Carvacrol decreased the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic F I G U R E 1 Carvacrol biosynthetic pathways via the mevalonate pathway. acid dehydrogenase (LDH) and improved the status of inflammation, necrosis, and coagulation in the liver (Bakır et al., 2016).
Carvacrol has anti-inflammatory characteristics and protects against digestive and oral disease by acting as an agonist of the TRPA1 receptor (transient receptor potential cation channel, subfamily A, member 1). In 2016, Alvarenga et al. found that carvacrol had anti-inflammatory property and helps us to maintain intestinal oral disease caused by irinotecan hydrochloride (CPT-11) 75 mg/kg, i.p. for 4 days via TRPA1 activation. Carvacrol activated TRPA1, decreased some inflammatory indicators such as MPO, NF-B, and COX-2 receptors; reduced the release or production of pro-inflammatory cytokines TNF-, IL-1, and KC, and lowered oxygen reactive species such as GSH, MDA, and NOx levels. Carvacrol also improved the blood bacterial count, leukogram, body mass variability, and survival rate, while also restoring villi structure in the small bowel (Alvarenga et al., 2016).
Another study conducted by Banik et al. in 2019 examined carvacrol's effects on apoptosis in PC12 cells induced by cadmium for 48 h in animals which increased the level of glutathione and glutathione reductase expression. Carvacrol also reduced the DNA fragmentation magnitude and also downregulated the level of mammalian target of rapamycin, protein kinase B (Akt), extracellular signal-regulated kinase-1, nuclear factor erythroid 2-related factor 2 (Nrf2) expressions, and nuclear factor kappa-light-chain-enhancer of activated B cells (NFКB). Carvacrol also reversed the action of cadmium via decreasing cytochrome c levels and also decreased the cleavage of caspase 3, an apoptosis-inducing factor. Carvacrol increased the intracellular metallothionein content (Banik et al., 2019).
The free hydroxyl functional group, specifically the molecular configuration, is the major contributing factor to its excellent antimicrobial capacity (Wang & Wu, 2021).
That is investigated that carvacrol increased the actions of anti-oxidant enzymes and decreased lipid peroxidation levels in the liver. Carvacrol inhibits the liver damage caused by aging as it reduced oxidative stress in animal models (Samarghandian, Azimi-Nezhad et al., 2016;. Arigesavan and Sudhandiran (2015) showed the anti-oxidant property of carvacrol extracted from O. vulgare sp. and assessed its effect on colitis-associated colon cancer (CACC) in male Fischer 344 rats. Carvacrol (50 mg) before and after tumor induction increased anti-oxidant enzymes such as catalase, superoxide dismutase, and glutathione levels, reduced (myeloperoxidase, lipid peroxides, and nitric oxide), and restored the histological lesions in the colitis.
Carvacrol also suppressed inducible nitric oxide synthase (iNOS) and pro-inflammatory mediators such as interleukin-1 beta (IL-1β) (Arigesavan & Sudhandiran, 2015; Figures 2 and 3). Rezvi & Roy, 2019). Its antiproliferative activities induce apoptosis, which further increases the expression of pro-apoptotic proteins. In cancer cells lines JAR and JEG3 cells, carvacrol induces calcium ions burden in the mitochondrial matrix via disrupting the mitochondrial membrane, suppresses extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) signal transduction, also decreases the phosphoinositide 3-kinase/protein kinase B, and increases phosphor-P38 and c-Jun N-terminal kinase MAPK expressions (Chraibi et al., 2020;Lim et al., 2019). Carvacrol in normal cells (L92) was also found to induce apoptosis via mitochondrial membrane potential disruption, ROS generation, activation of caspase3, and DNA damage (Jamali et al., 2020). A study conducted by Fan et al. explored that carvacrol works as an anticancer agent in different human colon cell lines such as LoVo and HCT116 via decreasing the matrix metalloprotease 2 and 9, cell-proliferation, cyclin B1 expression, and In causing In cell In cycle arrest at G2 and M phases. Additionally, it also increased phosphorylation of the extracellular-regulated protein kinase B and downregulated Bcl-2 expression (Fan et al., 2015). It also exhibited the dose-dependent inhibition in tumor growth cells in prostate cancer cells (PC3 cells) (Trindade et al., 2019). In a study, carvacrol-encapsulated nanoemulsion (CEN) formulated by a combination of polysorbate 80, lecithin, and MCT in lung A549 cells line in a dose-dependent manner reduced the activation of MAPK, p38, and ERK and decreased the expression of CD31 and VEGF (Carvalho et al., 2020;Khan, Bhardwaj, Shukla, Lee, et al., 2019;Khan, Bhardwaj, Shukla, Min, et al., 2019;Khan, Singh et al., 2019).

| Anticancer activity
Carvacrol can inhibit prostate cancer progression by inducing programmed cell death and cell cycle arrest at G0 and M-phases. In a dose-and time-dependent manner, carvacrol exhibited protective effects against prostate cancer cells via lowering cell viability, increasing the rate of reactive oxygen species, and disrupting the mitochondrial membrane potential. Carvacrol induced cell cycle arrest at G0/G1 that declined increased CDK inhibitor p21 expression and decreased cyclin-dependent kinase 4 (CDK4), and cyclin D1 expressions. Moreover, carvacrol inhibited Notch signaling in PC-3 cells via downregulating Jagged-1 and Notch-1 (Karam et al., 2019).
Heidarian examined the dose-dependent effects of carvacrol in human prostate cancer cell lines, which significantly reduced IL-6 gene expression as compared to the control group in which IL-6 protein reduced 41.5% and 52.7% at 360 and 420 μM. Carvacrol reduced cellular signaling proteins and gene expression and cellular signaling proteins. Further, it also caused a reduction in the cell survival rate, invasion, and proliferation rate (Bayir et al., 2019;Heidarian & Keloushadi, 2019). A study reported by Pakdemirli et al. in 2020 examined carvacrol effects on both HT-29 and HCT-116 via lowering the survival rate and proliferation rate (Pakdemirli et al., 2020).
In different in vitro and in vivo studies of MDA-MB 231 cells, carvacrol-induced apoptosis lowered the mitochondrial membrane potential resulting in the release of cytochrome c from mitochondria, cleavage of PARP, and caspase activation (Arunasree, 2010).
Carvacrol has significant protective effects in reducing the side effects of chemotherapeutics such as irinotecan hydrochloride anticancer drugs that cause induction of intestinal mucositis. Irinotecan hydrochloride triggers inflammation and leads to cell-damaging by the transient receptor potential cation channel, subfamily A, and member 1 receptor. Carvacrol reduced inflammatory biomarkers, such as nuclear factor κB and cyclooxygenase-2, and levels of Nitric oxides, malondialdehyde, and glutathione create oxidative stress.
It also acts as an agonist of the transient receptor potential cation channel (Alvarenga et al., 2016). In human cervical cancer HeLa cells, Potočnjak studied the anticancer role of carvacrol against human cervical cancer HeLa cells via decreasing the cell viability, inducing apoptosis, and inhibiting the mitogen-activated protein kinase (Potočnjak et al., 2018;Zeytun & Özkorkmaz, 2021).
Carvacrol was found to have antitumor, antiproliferative, and apoptotic activity against human colon cancer cell lines LoVo and HCT116 when combined with thyme (Fan et al., 2015). Carvacrol decreased cancer cells proliferation and apoptosis via decreasing matrix metalloprotease (MMP-2, MMP-9) expressions while downregulating the Bcl-2 expression and inducing phosphorylation of F I G U R E 2 Anti-oxidant potential of carvacrol. extracellular regulated protein kinase and protein kinase B(p-Akt) at the molecular level (Fan et al., 2015).

| Antidiabetic role
Diabetic cardiomyopathy is characterized by an abnormality in diastolic relaxation and later heart failure without hypertension, dyslipidemia, and coronary artery disease. Hyperglycemia, insulin resistance, and increased insulin levels in the case of diabetes lead to the development of diabetic cardiomyopathy (Jia et al., 2018;Liu et al., 2020). Carvacrol restored PI3K/AKT signaling-mediated GLUT4 membrane translocation and modulation of the PI3K. A study investigated the antidiabetic effects of intraperitoneal administration of carvacrol on streptozotocin-generated type-1 diabetes mellitus and type-2 diabetes mellitus in db/db mouse model for 6 weeks. Carvacrol significantly improved blood glucose levels, cardiac fibrosis and reversed cardiac hypertrophy, Myh7, and Nppa F I G U R E 3 Health perspectives of carvacrol.

22
carvacrol Improves metabolism and digestion ANTICANCER ACTIVITY Induce pro-apoptosis and reduce matrixexpression Metalloprotease 2, 9 in melanoma cell of gastric, colon, larynx, acute and chronic leiomyosarcoma cells.
Induce cell cycle arrest at G0 and G1 phases.

| Anti-obesity effects
De novo lipogenesis is the process of formation of new adipose cells derived from the ChREBP transcription factor. In this process, Carvacrol can control obesity by inhibiting intracellular fat accumulation and adipocyte differentiation as evidenced in high-fatdiet-induced male C57BL/6N mice embryo 3T3-L1 cells and the mechanism involved in gene expression in thermogenesis, adipogenesis, and inflammation. Carvacrol inhibited visceral adipogenesis through suppression of bone morphogenic protein-, galanin-mediated signaling, and fibroblast growth factor-1. Carvacrol inhibited toll-like receptor (TLR2 and TLR4)-mediated signaling and improved proinflammatory cytokines formation in visceral adipose tissues (Cho et al., 2012). Carvacrol in combination with rosiglitazone on diabetic mice C57BL/6J showed a reduction in triglycerides, low-density lipoproteins cholesterol, total cholesterol, phospholipids, and free fatty acids (Ezhumalai et al., 2015). A study conducted by Umaya and Manpal found that carvacrol has an anti-obesity role on embryo 3T3-L1 cells via lowering fat deposition in cells and visceral fats and also improving free fatty acids, liver cholesterol, and HDL-cholesterol.
Carvacrol reduced adipogenesis-related gene fibroblast growth factor receptor in visceral adipose tissues and galanin receptor 1 and 2 expressions (Suganthi & Manpal, 2013). In a high-fat-induced C57BL/6J mice study, carvacrol (20 mg/kg BW) used in combination with thiazolidinediones and rosiglitazone lowered the plasma glucose level, increased hemoglobin level, and increased as well as also alanine aminotransferase, alkaline phosphatase, and aspartate aminotransferase and gamma-glutamyl transpeptidase (Ezhumalai et al., 2014). In addition, carvacrol (25 and 50 mg/kg) was supplemented to streptozotocin-induced diabetes rats for 7 days and found that carvacrol significantly reduced the level of glucose, serum total cholesterol, and body weight changes (Amiri & Akbari, 2018).

| Pain management
A study conducted by Guimarães and fellows showed the carvacrol interaction with IL-10 and GABAA in which carvacrol 12.5-50 mg/kg once daily for 15 days in mice that significantly decreased mechanical hyperalgesia, improved use paw, spontaneous and palpation-induced nociception. Carvacrol also decreased neurons in the lumbar spinal cord, nucleus raphe Magnus and locus coeruleus, as well as activated peri-aqueductal gray (Guimarães et al., 2014). Guimarães et al. (2014) investigated that carvacrol has been used to cure pain caused by inflammation and mechanical hyper-nociception. Carvacrol is also associated with reducing pains in intraoral and oro-facial structures such as the mouth, face, head, and neck .
Likewise, administrated carvacrol at the rate of 100 mg/kg exhibited antinociceptive effects via lowering the inflammatory mediators and pain (Milovanović et al., 2016).

| Anti-inflammatory effects
In asthma, airway inflammation can be suppressed by peroxisome proliferator-activated receptor-alpha (PPARα) agonists as it reduces the release of inflammatory mediators majorly involved in asthma Rolim et al., 2019;Sun et al., 2020). Carvacrol protects from intestinal mucositis as it acts as an agonist of transient receptor potential cation channel, subfamily A, member 1(TRPA1).

| Neuroprotective role
The neuroprotective role of carvacrol was examined by Guan et al.

| Hepatoprotective and gastroprotective role
Carvacrol protects from hepatotoxicity caused by D-galactosamine It also acts as an agonist of the transient receptor potential cation channel. Carvacrol also restored the tissue architecture of the villi and crypts in the small intestine and side by side improved the blood bacterial count, leukogram, body mass variation, and survival rate (Alvarenga et al., 2016). Arigesavan and Sudhandiran (2015) showed the anti-inflammatory effects of carvacrol in the colon of Fischer 344 rats against inflammation induced by 1, 2-dimethyl hydrazine plus dextran sodium sulfate. F344 rats were given three subcutaneous injections of DMH (40 mg/kg body wt) in the first week to F344 rats and free access to drinking water containing 1% DSS for the next 1 week was also given for 7-14 days as three cycles. 50 mg/kg body weight (o.p) carvacrol was administrated before and after tumor induction. Carvacrol-treated groups suppress the inflammation in DMH/DSS-induced animals, increased anti-oxidant status; developed an endogenous anti-oxidant system was observed and restorative histological lesions. Carvacrol also increased significantly the level of anti-oxidant enzymes such as glutathione levels, superoxide dismutase, catalase, reduced nitric oxide, lipid peroxides, interleukin-1 beta, and myeloperoxidase as compared to DMH/DSS induced rats (Arigesavan & Sudhandiran, 2015).

| Cardioprotective
Carvacrol works as an agonist/antagonist of different voltagedependent calcium channels and TRP channels due to their involvement in bradycardia and peripheral vasodilation (Dantas et al., 2015). type male C57 BL/6 mice prevents myocardial ischemia-reperfusion injury via lowering myocardial infarct size, oxidative stress level, and cardiac myocyte apoptosis rate . Lead is known to cause constriction of heart vessels, whereas Shabir found contracting response of aortal rings of rats induced by Pb(II) via elevation of ROS and depletion of nitric oxide. Carvacrol treatment prevented these contracting responses of aortal rings in rats (Shabir et al., 2014). Similarly, cyclophosphamide increased the serum alanine transaminase, aspartate transaminase, lactate dehydrogenase, creatine kinase-MB, total oxidant state, and oxidative stress index in experimental animals, whereas different concentrations of carvacrol at 5.0 and 10 mg/kg reverted these changes (Cetik et al., 2015).
Left ventricular hypertrophy is caused by cardiomyocytes apoptosis induced by Bcl-2 family members involved in regulating mitochondrial pathways of apoptosis. Carvacrol has a positive impact on the transcription level of antiapoptotic (Bcl2 and Bcl-xL) members and pro-apoptotic (Bad and Bax) of Bcl-2 family in hypertrophied hearts of male Wistar rats as well as lowered heart weight to body weight ratio, and Bad mRNA level (Sadeghzadeh et al., 2018).
Carvacrol inhibited platelet-derived growth factor (PDGF)-BB which stimulated aortic smooth muscle cell proliferation and migration dose-dependently as well as decreased aortic sprout outgrowth and reduced the phosphorylation of MAPK and ERK1/2 (Lee et al., 2015).
A study conducted by Jamhiri and fellows found that different in vitro and in vivo administrations of carvacrol (25, 50, and 75 mg/kg/ day) against left ventricular hypertrophy of male Wistar rats reduced blood pressure, heart rate, and heart weight to the body weight ratio. In the in vitro study, carvacrol momentously lowered the H9c2 cell size as compared to Ang II-treated cells. Furthermore, 50 and 75 mg/kg/day doses significantly inhibited the atrial natriuretic peptide and lowered the number of apoptotic cells (Alves et al., 2016). It significantly caused a reduction in absolute eosinophil count, absolute eosinophil count, immunoglobulin E, inflammatory biomarkers (TNFα, L-4, IL-5, IL-13, and interferon-gamma), and enhancement in anti-oxidant enzymes further that prevent from the inflammatory symptoms in asthma (Ezz-Eldin et al., 2020). Combination of carvacrol with essential oils (Foeniculum vulgare, Saturea cuneifolia, and Origanum munitiflorum) can inhibit ROS production, T-cells proliferation, and pro-inflammatory cytokines (Khazdair et al., 2018;Orhan et al., 2016). Encapsulated carvacrol (250-650 μg/g) in necrotic enteritis animal disease caused by C. perfringens in chicken intestine prevented the immune-mediated responses (Liu et al., 2016). Thymol and carvacrol in human mesenchymal stromal cells protect from oxidative stress-related damage and cytotoxicity and preserve cell morphology (Bouhtit et al., 2019).  (Gholijani et al., 2015). A study reported by Gandhi et al. found that carvacrol also regulates cytokine production, inhibits ROS accumulations, and inactivates eosinophils migration lungs. EO suppressed cytokine production, pro-inflammatory and anti-inflammatory mediators formation, and accumulation (Gandhi et al., 2020).

Tolerogenic dendritic cells (DCs
Carvacrol also protects in case of multiple sclerosis ( (Türkcü et al., 2015). Carvacrol effects on cytokines genes expression in splenocytes of asthmatic mice were studied by Kianmehr et al. (2016) in rats in which asthma is induced by ovalbumin (OVA) and it was concluded that carvacrol significantly modulated the immune response by decreasing IL-4, IL-17, and TGFβ gene expressions and increased IFNγ and FOXP3 (Kianmehr et al., 2016). Boskabady (2019a, 2019b) investigated carvacrol effects on serum levels of interferon-gamma (IFNγ), interleukins (IL-2, IL-4, IL-6, IL-8, and IL-10), and pulmonary function tests in 22 patients exposed to sulfur mustard (SM) 27-30 years in a double-blind manner for 2 months divided into placebo and carvacrol 1.2 mg/ kg/day. It was concluded that carvacrol reduced inflammatory cytokines, while increased anti-inflammatory cytokines and improved PFT tests in SM-induced lung injury (Khazdair & Boskabady, 2019a. Another study evaluated the immunomodulatory and ulcer protective action of carvacrol (25, 50 & 100 mg/kg) on an animal model in which gastric lesions were made by acetic acid. Results showed carvacrol gastric healing actions and also proved that it interferes with secretion and production of inflammatory mediators in case of ulcer (Hussein et al., 2019;Silva et al., 2012; Table 1). at the levels of gene and protein expression (Shoorei et al., 2019).

| REPRODUC TIVE ROLE
A study conducted by Güvenç et al. (2019) found that carvacrol in combination with thymol has a significant impact on the quality of sperms improved by decreasing level of oxidative stress, MDA levels in testicles, liver, and kidney tissues, enhancing the GSH-Px and catalase activities along with enhancement in spermatozoa concentration and motility (Güvenç et al., 2019). Moreover, carvacrol also improves the mean motility, movement characteristics, sperm capacitation, and fertilizing ability and prevents testicular damage . Carvacrol (25 and 50 mg/kg) prevented ketamine-induced oxidative stress and damage in testicular tissues by lowering the level of MDA-induced schizophrenia and increasing the anti-oxidant enzymes (Araghi et al., 2017). Similarly, carvacrol prevents cyclophosphamide-induced testis toxicity and damage in male rats due to its anti-oxidative role . In adult male Sprague-Dawley rats, cisplatin induces reproductive toxicity by damaging the dermatological parameters (live sperm rate, motility, and abnormal sperm rate), increasing the oxidative stress, and TA B L E 1 Carvacrol's health benefits and method of action

Health perspective Mechanism of action References
1 Anti-oxidant activity Hepatoprotective effects DNA protection Anti-oxidant activity mediates anticancer effects Increase in anti-oxidant defense leads to improved immune system response Reduction of oxidative stress damage in the brain, liver, and kidney of rats Alagawany et al. (2015) and Sun et al. (2020) 2 Anticancer activity Cytotoxic, genotoxic, and proapoptotic activities with effects on cell invasion by decreasing the expression of matrix Metalloprotease 2 and 9 (melanoma cell, larynx, colon, gastric, leiomyosarcoma cells, and chronic myeloid leukemia, cells), K562, A549 non-small-cell lung cancer cells, MCF-7, and MDA-MB-231 human metastatic breast cancer cells Genomic DNA fragmentations and caspase-3, caspase-6, or caspase-9 enzymes gene expression were induced by carvacrol; also carvacrol induces apoptosis regulatory genes in human cancer and retarded growth Zeytun and Özkorkmaz (2021) 3 Antidiabetic role Carvacrol significantly restored PI3K/AKT signaling, which was impaired in mice with T1DM and T2DM. Carvacrol increased levels of phosphorylated PI3K, PDK1, AKT, and AS160 and inhibited PTEN phosphorylation in mice with T1DM and T2DM. Carvacrol treatment promoted GLUT4 membrane translocation in mice with T1DM and T2DM Bayramoglu et al. (2014) and Hou et al. (2019aHou et al. ( , 2019b 4

Anti-inflammatory effects
Carvacrol acts as agonist of the TRPA1 receptor Reduction in the production or release of pro-inflammatory cytokines (TNFα, IL-1β, and KC) and decrease in indicators of inflammation (MPO, NF-κB, COX-2) and oxidative stress (GSH, MDA, and NOx levels) Alvarenga et al. (2016) 5 Hepatoprotective effect Protects liver during renal injury and hepatic injury by improving liver anti-oxidant defense and minimizing the products of lipid peroxidation Decrease TNFα levels in pleural lavage Decrease the levels of enzyme responsible for inducing nitric oxide synthase Bakır et al. (2016) 6 Neuroprotective role Carvacrol provides protection for hippocampal neurons against I/R in gerbils by inhibiting ferroptosis through increasing the expression of GPx4

| Safety and toxicity
Carvacrol is considered the safest chemical compound at a low amount, approved by Federal Drug Administration (FDA), and used as a preservative in the food industry (Suntres et al., 2015). Ghorani et al. (2021) designed a study to determine the carvacrol tolerability and safety in normal-healthy individuals by dividing them into two groups having 1-2 mg of carvacrol per body weight a day.
In the group received 1 mg/kg/day of carvacrol, their erythrocyte sedimentation rate, mean cell volume, hemoglobin, and hematocrit levels were reduced, but creatinine phosphokinase was significantly increased. Their triglyceride, phosphorus, lactate dehydrogenase, prothrombin time, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly increased after treatment with carvacrol 1 mg/kg/day. In the group received 2 mg/ kg/day of carvacrol, their high-density lipoprotein cholesterol (HDL), total bilirubin, amylase, iron, red blood cells (RBC) count, and HCT reduced after one-month treatment (Ghorani et al., 2021).

| CON CLUS ION
The purpose of this comprehensive review was to highlight and explain the biosynthesis mechanism of carvacrol and the significant protective effects of carvacrol as an anti-oxidant, anticancer potential, anti-inflammatory, antimicrobial, hepatoprotective effects, and neuro-protective and anti-obesity effects. Scientists and researchers have studied the potential of carvacrol and thymol for cancer prevention, diabetes prevention, wound healing, and immuno-modulatory in vivo and in vitro assays. Carvacrol exhibits a high potential for the development of new therapeutic alternatives to cure human maladies. However, the extensive studies still required to elucidate the potential therapeutic effect of carvacrol on molecular level by involving long-term human efficacy trial with special reference to its lethal dose, toxicity, and RDA.

ACK N OWLED G EM ENT
The authors acknowledge HEC for its support.